The urinary tract is among the most common sites of bacterial infection and E. coli is by far the most common species infecting this site. Most data regarding expression of uropathogenic E. coli (UPEC) virulence genes have come from in vitro studies or the murine model of urinary tract infection (UTI). To remedy this, we have measured global gene expression from E. coli in urine collected and stabilized immediately from women presenting with uncomplicated UTI. These new RNAseq data allow us to focus on the most highly expressed virulence mechanisms active during human infection. Our long term research goal is to understand how UPEC colonize the human urinary tract, avoid the immune response, and damage the host. The objective during this funding period is to determine the contribution to pathogenesis of the virulence genes found to be specifically upregulated in the human host. Our central hypothesis, that the virulence of a UPEC strain is the sum of its capacity for adherence, iron acquisition, toxin production and specific metabolite transport, is supported by our measurements of global gene expression patterns of UPEC during UTI in five women attending the University Health Service Clinic at the University of Michigan for the symptoms of cystitis. The rationale for the proposed work is that once we identify the most highly expressed virulence genes during UTI in humans, we can focus efforts on intervention and prevention directed toward these specific targets. We will test our central hypothesis and complete our objectives by carrying out two specific aims: 1) Determine the representative transcriptome for uropathogenic E. coli during urinary tract infection in humans; and 2) Determine the mechanism of pathogenesis for newly discovered virulence determinants upregulated in E. coli infecting women with urinary tract infection. The expected outcomes of conducting these aims will be a precise assessment of global gene expression and host-specific virulence genes upregulated during infection in women, but not during in vitro culture in urine or rich medium. The positive impact of these studies will be substantial. We will precisely quantify UPEC gene expression during infection of otherwise healthy women. We will determine the mechanisms of action of the newly identified virulence factors active during infection of the urinary tract by E. coli. We will also understand which virulence determinants are both present and expressed during uncomplicated human UTI, and the degree to which they are expressed. Understanding the virulence factors used by the bacterium to colonize the human urinary tract, avoid the immune response, and damage the host will open the door to preventing this public health scourge in women with recurrent UTI and those susceptible to their first UTI.